Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G

Mitsunori Shiroishia,b, Kouhei Tsumotob, Kimie Amanoc, Yasuo Shirakiharac, Marco Colonnad, Veronique M. Braude,f, David S. J. Allane, Azure Makadzangee, Sarah Rowland-Jonese, Benjamin Willcoxg, E. Yvonne Jonesg, P. Anton van der Merweh, Izumi Kumagaib, and Katsumi Maenakaa,c,i

aDivision of Structural Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; bDepartment of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-yama 07, Sendai 980-8579, Japan; cStructural Biology Center, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan; dDepartment of Pathology and Immunology, Washington University School of Medicine, Box 8118, 660 South Euclid Avenue, St. Louis, MO 63110; eNuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom; gCancer Research U.K. Receptor Structure Group, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Headington, Oxford OX3 7BN, United Kingdom; and hSir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom

Edited by Max D. Cooper, University of Alabama at Birmingham, Birmingham, AL, and approved May 20, 2003 (received for review February 23, 2003) Ig-like transcript 4 (ILT4) (also known as leukocyte Ig-like receptor nization (9). Studies on CD8ϩ cells suggest that ILT2 is ex- 2, CD85d, and LILRB2) is a cell surface receptor expressed mainly on pressed early on in contrast to KIRs, which are expressed myelomonocytic cells, whereas ILT2 (also known as leukocyte primarily on the subset of stimulated CD8ϩ cells that become Ig-like receptor 1, CD85j, and LILRB1) is expressed on a wider range long-term memory cells (10). In addition, ILT2 is a major of immune cells including subsets of natural killer and T cells. Both inhibitory receptor on NK cells for the nonclassical HLA-G ILTs contain immunoreceptor tyrosine-based inhibitory receptor ligand. On the other hand, ILT4 may be involved in regulating motifs in their cytoplasmic tails that inhibit cellular responses by the activation of inflammatory cells in a range of systems: (i) recruiting phosphatases such as SHP-1 (Src homology 2 domain CD68ϩ macrophages and neutrophils in synovium from rheu- containing tyrosine phosphatase 1). Although these ILTs have been matoid arthritis patients (11); (ii) myelomonocytic cells in ma- shown to recognize a broad range of classical and nonclassical terno-fetal tolerance (12); and (iii) tolerogenic antigen- human MHC class I molecules (MHCIs), their precise binding prop- presenting cells induced by regulatory T cells (13). erties remain controversial. We have used surface plasmon reso- Based on killing and cell-binding assays, ILT2 and ILT4 have nance to analyze the interaction of soluble forms of ILT4 and ILT2 been shown to bind to classical (HLA-A and -B) and nonclassical with several MHCIs. Although the range of affinities measured was (HLA-G1, -E, and -F) MHC class I molecules (MHCIs). In these ␮M), some interesting differences were 45–2 ؍ quite broad (K d cellular assays, minimal or no binding to HLA-C was detected observed. ILT2 generally bound with a 2- to 3-fold higher affinity (3–5, 7, 8, 12, 14–16). Cellular assays also indicate that ILT2 than ILT4 to the same MHCI. Furthermore, ILT2 and ILT4 bound to HLA-G with a 3- to 4-fold higher affinity than to classical MHCIs, binds to the MHCI homolog from human cytomegalovirus suggesting that ILT͞HLA-G recognition may play a dominant role in UL18, whereas ILT4 does not. Using purified proteins, however, the regulation of natural killer, T, and myelomonocytic cell activa- Chapman et al. (17) detected ILT2 binding to HLA-C alleles and tion. Finally, we show that ILT2 and ILT4 effectively compete with ILT4 binding to UL18, with affinities that are within the range CD8 for MHCI binding, raising the possibility that ILT2 modulates typical of cell–cell recognition interactions. -CD8؉ T cell activation by blocking the CD8 binding as well as by Domain deletion and mutational analyses of the ILT2 ectodo recruiting inhibitory molecules through its immunoreceptor ty- main have revealed that the N-terminal domain 1 (D1) is the rosine-based inhibitory receptor motif. main MHCI-binding region (17, 18). Conversely, domain- swapping experiments have shown that ILTs bind to the ␣3 leukocyte Ig-like receptors ͉ major histocompatibility complex ͉ domain of MHCIs. This is distinct from the KIR-binding site, surface plasmon resonance ͉ natural killer cell ͉ coreceptor which lies within the ␣1–␣2 region (17, 19, 20). Because the ␣3 domain is relatively more conserved among the MHCI alleles ␣ ␣ g-like transcripts (ILTs) (also called leukocyte Ig-like recep- than the polymorphic 1– 2 peptide-binding region, these data Itors, CD85, or LILRB) are encoded by a family of immuno- account for the broader binding specificity of ILT2 compared receptor genes located at human chromosome 19q13.4. This with KIRs. The crystal structure of the D1 and D2 region of ILT2 locus is called the leukocyte receptor complex and includes, in (ILT2D1D2) has been solved (18), demonstrating that addition to ILT genes, the genes encoding killer cell Ig-like ILT2D1D2 had two Ig-like domains in tandem related by an receptors (KIRs), leukocyte-associated Ig-like receptors, acute elbow angle, similar to KIRs. However, the MHC-binding NKp46, and the Fc␣ receptor (1). Although ILT2 is broadly sites are distinct. The binding site on ILT2 is confined to the GFC expressed on monocytes, B cells, dendritic cells, and subsets of ␤-sheet surface of the D1 domain [Ig domains possess two natural killer (NK) and T cells, ILT4 expression is largely ␤-sheets, made up of ABE(D) or GFC(CЈCЉ) ␤-strands, respec- confined to the myelomonocytic lineage (2–8). Both ILT2 and ILT4 have four tandem Ig-like extracellular domains and four and three immunoreceptor tyrosine-based inhibitory receptor This paper was submitted directly (Track II) to the PNAS office. motifs, respectively, in their cytoplasmic tails. Immunoreceptor Abbreviations: ILT, Ig-like transcript; KIR, killer cell Ig-like receptor; NK, natural killer; TCR, ␤ tyrosine-based inhibitory receptor motifs recruit the protein T cell antigen receptor; MHCI, MHC class I molecule; Dn, N-terminal domain n; 2m, ␤ -microglobulin; SPR, surface plasmon resonance. tyrosine phosphatase SHP-1 (Src homology 2 domain containing 2 fPresent address: Institut de Pharmacologie Moleculaire et Cellulaire, Centre National de la phosphatase 1), which is thought to inhibit early signaling events Recherche Scientifique, Sophia Antipolis 06560, France. triggered by stimulatory receptors. Indeed engagement of ILT2 iTo whom correspondence should be addressed at: Division of Structural Biology, Medical on T cells has been shown to inhibit T cell antigen receptor Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812- (TCR) signaling and downstream events such as actin reorga- 8582, Japan. E-mail: [email protected].

8856–8861 ͉ PNAS ͉ July 22, 2003 ͉ vol. 100 ͉ no. 15 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1431057100 Downloaded by guest on September 25, 2021 tively], whereas the corresponding site on KIRs involves the reaction buffer. After biotinylation, MHCIs were separated interdomain region between domains 1 and 2. from the reaction mixture by gel filtration (Superdex 75). In contrast to ILT2, the binding studies on recombinant ILT4 Biotinylated HLA-Cw*0702 was refolded with peptide [DS11 have been limited to the ILT4–UL18 interaction (18). Here we (RYRPGTVAL) or DS12 (NKADVILKY)] and chemically report an investigation of the binding of ILT4 to a wide range of biotinylated ␤2-microglobulin (␤2m) in the same way as other classical and nonclassical MHCIs, together with a thorough MHCI complexes. Purification was accomplished by using Su- analysis of ILT2 binding. ILT4 and ILT2 bound with low perdex 200 and MonoQ columns. affinities to all MHCIs tested. Unexpectedly, ILT4 and ILT2 bound to the nonclassical MHCI HLA-G with 3- to 4-fold higher Surface Plasmon Resonance (SPR). SPR experiments were per- affinity than to classical MHCIs. Because the HLA-G molecule formed by using a BIAcore2000 (BIAcore, St. Albans, U.K.). is uniquely expressed on the immunologically relevant sites such The biotinylated MHCIs and control protein (biotinylated OX48 as trophoblasts in placenta, thymic epithelial cells, and some antibody or BSA) were immobilized on the research-grade CM5 tumors (21–25) including glioma cells (26), this result suggests an chip (BIAcore), onto which streptavidin was covalently coupled. important in vivo role for ILT–HLA-G interactions. Finally, ILT2D1D2 and ILT4D1D2, after buffer exchange to HBS-EP competition assays showed that ILTs compete with CD8 for (10 mM Hepes, pH7.4͞150 mM NaCl͞3.4 mM EDTA͞0.005% binding to MHCI. We discuss the implications of these findings Surfactant P20), were injected over the immobilized MHCIs. for the function of ILTs on CD8ϩ T cells and NK cells. The binding response at each concentration was calculated by subtracting the equilibrium response measured in the control Materials and Methods flow cell from the response in the MHCIs flow cell. Kinetic Production of ILT2 and ILT4 Ectodomains. DNA encoding the first constants were derived by using the curve-fitting facility of the two extracellular domains (residues 1–197) of ILT4 was ampli- BIAEVALUATION 3.0 program (BIAcore) to fit rate equations fied from cDNA (6) by using 5Ј-G GAA CAT ATG GGG ACC derived from the simple 1:1 Langmuir binding model (A ϩ B ↔ ATC CCC AAG CCC-3Ј as forward primer and 5Ј-CC CAA AB). Other curve fitting was performed by ORIGIN 3 (Microcal GCT TAC TAT GGG ACC AGG AAG CTC CAG G-3Ј as Software, Northampton, MA). Affinity constants (Kd) were reverse primer. The resultant fragments were digested with the derived by Scatchard analysis or nonlinear curve fitting of the restriction enzymes NdeI and HindIII and ligated into the standard Langmuir binding isotherm. pGMT7 vector (27) (designated pGMILT4D1D2). Escherichia The whole ectodomain of ILT2 fused with Fc was indirectly coli strain BL21(DE3)pLysS cells (Novagen) harboring coupled to the SPR sensor surface by using an anti-human-Fc pGMILT4D1D2 produced ILT4D1D2 inclusion bodies. They mAb. Sensor flow-cell surfaces coated with mAb alone served as were isolated from cell pellet by sonication and washed repeat- paired controls. With regards to ILT4, the whole ectodomain was edly with wash solution including 0.5% Triton X-100. The DNA coupled indirectly to the SPR sensor chip by using the anti-ILT encoding ILT2D1D2 region (residues 1–197) designed by using 40H2 mAb. MHCIs were injected over the immobilized ILTs in E. coli-favored codons was constructed by PCR with 10 chem- HBS-EP. ically synthesized DNAs (see Table 2, which is published as supporting information on the PNAS web site, www.pnas.org). Competitive Binding Assays. The ectodomain of KIR2DL1 (resi- The fragment was inserted into the pGMT7 vector (designated dues 1–224) and CD8␣ homodimer (CD8␣␣) (residues 1–120) pGMILT2D1D2), and inclusion bodies containing ILT2D1D2 were produced as described (27, 29). ILT2D1D2 and ILT4D1D2 were obtained by using the same method as described above. with or without a fixed amount of KIR2DL1(38 ␮M), which was The purified ILT2D1D2 inclusion bodies were solubilized in almost saturated, were flowed over the immobilized HLA- denaturant solution including 6 M guanidine hydrochloride. By Cw*0401. ILT2D1D2 and ILT4D1D2, with or without a fixed using the refolding buffer (0.1 M Tris⅐HCl, pH 8.0͞0.4 M concentration of CD8␣␣ (92 ␮M), were flowed over the immo- L-arginine͞2 mM EDTA͞5 mM reduced glutathione͞0.5 mM bilized HLA-B*3501, -Cw*0401, and -G1. oxidized glutathione͞0.1 mM PMSF), the solubilized protein solution was diluted slowly to the final protein concentration of Results and Discussion 1–2 ␮M and stirred for 48 h at 4°C. Then the refolding mixture Production of Soluble ILTs and MHCIs. We initially attempted to of ILT2D1D2 or ILT4D1D2 was concentrated with a VIVA- express three forms of the ILT4 ectodomain: domain 1 alone FLOW50 system (Sartorius). ILT2D1D2 was purified by gel (D1, residues 1–98); domains 1 and 2 (D1D2, residues 1–197); filtration with Superdex 75 (Amersham Pharmacia). In the case and the entire extracellular domains (D1–D4, residues 1–435). of ILT4D1D2, after concentrating to 5 ␮M, the buffer was These constructs were expressed in E. coli as inclusion bodies exchanged gradually to 50 mM phosphate buffer, pH 6.0, with and subsequently refolded in vitro. ILT4D1 and ILT4D1–D4 the VIVAFLOW50 system. ILT4D1D2 was purified by anion- were difficult to refold and aggregated readily, making it im- exchange chromatography (SP Sepharose, Amersham Pharma- possible to produce enough material for further study. However, cia) followed by gel filtration (Superdex 75). ILT4D1D2 was successfully refolded and purified as described in The whole ectodomain of ILT2 was obtained from transfected Materials and Methods. Because stored ILT4D1D2 tended to J558L cells producing chimeric ILT2 molecule fused with the Fc aggregate, we prepared freshly refolded and purified ILT4D1D2 portion of human IgG1 (6). On the other hand, for the whole for each experiment. The equivalent ILT2 fragment ectodomain of ILT4, an expression system producing soluble (ILT2D1D2) was prepared by using a very similar method (see extracellular domains of ILT4 tagged at the C terminus with a Materials and Methods). The far-UV circular dichroism spectrum c-Myc tag and poly-His tail was constructed. of ILT4D1D2 indicated that it had mainly ␤-sheet secondary structure, similar to ILT2D1D2 and as expected for a protein Production of Soluble and Biotinylated MHC Molecules. Soluble with two Ig-like domains (ref. 18 and data not shown). For biotinylated HLA-A*1101 (with peptide AIFQSSMTK), HLA- comparison, soluble fragments comprising the entire ectodo- B*3501 (with peptide IPLTEEAEL), HLA-Cw*0401 (with main of ILT2 and ILT4 (ILT4D1–D4 and ILT2D1–D4) were peptide QYDDAVYKL), and HLA-G1 (with peptide produced by using the eukaryotic expression systems (see Ma- RIIPRHLQL) with C-terminal biotin ligase (BirA) recognition terials and Methods). sequence (GSLHHILDAQKMVWNHR) were prepared as de- All the MHCIs were expressed in bacterial inclusion bodies, scribed (27, 28). Each MHCI was biotinylated with 50 mM refolded in vitro with bacterially expressed ␤2m and appropriate IMMUNOLOGY D-biotin͞100 mM ATP͞15 ␮M BirA for 15 ␮M MHCI in the peptides, and purified as described in Material and Methods.With

Shiroishi et al. PNAS ͉ July 22, 2003 ͉ vol. 100 ͉ no. 15 ͉ 8857 Downloaded by guest on September 25, 2021 Fig. 1. Equilibrium binding of ILT2 and ILT4 to MHCIs. (A and C) ILT4D1D2 (35 ␮M) (A) and ILT2D1D2 (87 ␮M) (C) were injected for 30 s through flow cell 1 with control (BSA, solid line), flow cell 2 with HLA-B35 (broken and dotted line), flow cell 3 with HLA-Cw7 refolded by using chemically biotinylated ␤2m (broken line), and flow cell 4 with HLA-G1 (dotted line). Biotinylated BSA was used as a control. (B and D) Plots of the equilibrium binding responses of ILT4D1D2 (B) and ILT2D1D2 (D) versus concentration. Diamonds, HLA-A11; squares, HLA-B35; circles, HLA-Cw4; downward triangles, HLA-Cw7; upward triangles, HLA-G1. The solid lines represent direct nonlinear fits of the 1:1 Langmuir binding isoform to the data. (Insets) Scatchard plots of the same data are shown. The solid lines are linear fits. RU, response units.

the exception of HLA-Cw*0702, all MHC heavy chains incorpo- (Kd Ϸ 8.8 and 6.5 ␮M, respectively) similar to those measured for rated a C-terminal biotinylation tag (LHHILDAQKMVWNHR). KIR–MHCI interactions (Kd Ϸ 7–10 ␮M) (30, 32). As found with After refolding and purification they were biotinylated with biotin ILT4D1D2, the ILT2D1D2 bound with the highest affinity to ligase, which specifically biotinylates a lysine in the tag (underlined). HLA-G1 (Kd Ϸ 2.0 ␮M). As with ILT4, these results are HLA-Cw*0702 was refolded with chemically biotinylated ␤2m. consistent with the previous cellular-based assays with the Biotin-HLA-Cw*0702 produced in this way has been used success- exception of HLA-Cs. HLA-C transfectants do not bind effi- fully for KIR2DL3-binding studies (30). ciently to ILT2 Fc fusion protein or inhibit the killing activity of ILT2ϩ NK cells (6, 15). Affinities of ILT2 and ILT4 Binding Toward MHCIs. Affinity measure- The higher affinity of ILT2 and ILT4 for HLA-G1 versus other ments were performed by using SPR as implemented in the MHCIs (HLA-A2, -A68, -B8, and -E) was confirmed by using BIAcore2000 instrument. Soluble ILT4D1D2 or ILT2D1D2 was entire ectodomains (D1–D4) of both ILT2 and ILT4 produced injected over sensor surfaces to which biotinylated MHCI com- by eukaryotic cells (data not shown). In contrast, Chapman et al. plexes or (as negative controls) biotinylated OX48 antibody or BSA (17) reported that ILT2 binds HLA-G1 with a lower affinity had been immobilized. The affinities of ILT4D1D2 and ILT2D1D2 (Kd Ϸ 100 ␮M) than it binds other MHCIs. The reason for this binding to MHCIs were measured by equilibrium binding analysis. discrepancy is uncertain, but it may be the result of differences A range of concentrations of ILT4D1D2 or ILT2D1D2 was injected in the way MHCIs were immobilized. In our study we typically through flow cells with MHCIs immobilized. used the site-specific biotinylated tag at the C terminus of the Representative data for equilibrium binding of ILT4D1D2 to MHCI heavy chain, whereas Chapman et al. (17) randomly various MHCIs are shown in Fig. 1A. ILT4D1D2 binding coupled the MHCI via amines. The former method presents reached equilibrium and dissociated rapidly, indicating that it MHCI in a manner optimal for ligand binding, whereas it is bound MHCI with the fast kinetics that are typical of interac- possible that the random coupling of MHC disrupts or interferes tions mediating cell–cell recognition [for example, see Maenaka with ILT binding. et al. (30, 31)]. ILT2D1D2 binding also displayed fast kinetics (Fig. 1C and see below). Fitting of conventional and Scatchard ILTs Compete with CD8 for Binding to MHCIs. Although there is plots of ILT4D1D2 (Fig. 1B and Inset) and ILT2D1D2 (Fig. 1D evidence from domain-swapping experiments that ILT2 binds and Inset) binding data indicated that binding conformed to the the ␣3 domain of MHCI heavy chains (17), it is not known simple 1:1 (Langmuir) binding model. whether ILT binding interferes with the binding of other MHCI The results of multiple independent measurements for both ligands. We therefore used SPR to examine whether ILT binding ILT4D1D2 and ILT2D1D2 are summarized in Table 1 and Figs. to MHCI influenced the binding of the MHCI ligands KIR2DL1 4 and 5, which are published as supporting information on the and CD8 and the mAb BBM.1, which binds ␤2m. KIR2DL1 binds PNAS web site. ILT4D1D2 binds to a broad range of classical the region adjacent to the peptide C terminus on the peptide- MHCIs (HLA-A*1101, -B*3501, -Cw*0401, and -Cw*0702) with binding platform of HLA-Cw4 and related alleles [Fig. 2A and affinities (Kd) ranging from Ϸ14 to Ϸ45 ␮M (Fig. 5 and Table Fan et al. (20)]. 1). Interestingly, ILT4D1D2 binds with an even higher affinity Having established that KIR2DL1 binds HLA-Cw4 with an (Kd Ϸ 5 ␮M) to the nonclassical MHCI HLA-G1. A previous affinity of Kd Ϸ 3.3 ␮M (Fig. 2A), we examined the binding cellular binding study (7) reported that ILT4 binds to HLA-A2, response when increasing concentrations of ILT4 (Fig. 2B)or -A3, -B8, -B27, -B35, and -G1 but not to HLA-Cw3 or -Cw5. This ILT2 (Fig. 2C) were injected over HLA-Cw4 with (squares) or result is surprising given the small differences (one or two without (circles) a fixed, high concentration (38 ␮M) of residue changes) in the relevant (␣3) portions of HLA-Cw4, KIR2DL1. The difference between the responses remained the which we show binds ILT4 and HLA-Cw3 and -Cw5 (see Fig. same (crosses) whatever the concentration of ILTs, indicating 5C). However, there have been previous reports of discrepancies that the binding of ILTs and KIR2DL1 to HLA-Cw4 was purely between cell-based and direct binding studies when measuring additive. This demonstrates that the binding of ILTs does not ILT2–HLA-C interactions (6–8). The reason for these discrep- affect the binding of KIR2DL1 to the same molecule, consistent ancies remains unclear, but it may be related to avidity effects with previous data showing that they bind to different regions of (cell-based assays rely on multivalent binding) and͞or the low the MHCI. level of cell surface expression of HLA-C when compared with We next examined whether ILTs interfered with CD8 binding other MHC alleles. to MHCIs (Fig. 2 E–I). Having showed that soluble recombinant ILT2D1D2 binds to HLA-B35 and HLA-Cw4 with affinities CD8␣␣ bound to HLA-B35, -Cw4, and -G1 with affinities of

8858 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1431057100 Shiroishi et al. Downloaded by guest on September 25, 2021 Fig. 2. The effect of KIR2DL1, anti-␤2m mAb (BBM.1), or CD8␣␣ on the binding of a soluble ILT2 and ILT4 to MHCIs. (A) Equilibrium binding analysis of KIR2DL1 against HLA-Cw*0401 (Kd Ϸ 3.3 ␮M) on the same sensor chip used in the experiments shown in B and C. The estimated saturation level of KIR2DL1 was calculated by nonlinear curve fitting [1,026 response units (RU)]. (B and C) Binding of ILT4D1D2 (B) and ILT2D1D2 (C) (filled circles) alone or mixed with KIR2DL1 (filled squares). The concentration of KIR2DL1 was 38 ␮M(A, dotted line). The difference in the binding seen with or without KIR2DL1 was plotted (crosses). (D) Equilibrium binding analysis of CD8␣␣ against HLA-B*3501 (squares), HLA-Cw*0401 (circles), and HLA-G1 (upward triangles) on the same sensor chip used in the experiments shown in E–I. The Kd values are listed in Table 1. (E and F) Binding of ILT4D1D2 with or without CD8␣␣ to HLA-B*3501 and HLA-G1, respectively. The concentration of CD8␣␣ was 92 ␮M(D, dotted line). The difference in the binding seen with or without CD8␣␣ was plotted (crosses) in the experiments shown in E–I.(G–I) Binding of ILT2D1D2 with or without CD8␣␣ to HLA-B*3501 (G), HLA-Cw*0401 (H), and HLA-G1 (I). The concentration of CD8␣␣ was 92 ␮M. (J) Binding of ILT2D1D2 (105 ␮M), ILT4D1D2 (33 ␮M), and CD8␣␣ (92 ␮M) before and after injection of anti-␤2m BBM.1 mAb to saturation level.

Kd Ϸ 126, 210, and 72 ␮M, respectively (Fig. 2D and Table 1), The binding sites of KIR2DL1 (20), BBM.1 mAb (34), and we injected increasing concentrations of ILT2 and ILT4 in the CD8␣␣ (35), as defined by mutagenesis and structural studies, presence (squares) or absence (circles) of CD8␣␣ (92 ␮M) over are shown in Fig. 3C. Our results show that the ILT-binding site different MHCIs (Fig. 2 E–I). The difference between the overlaps the CD8␣␣-binding site (cyan surfaces in Fig. 3C). A responses with or without CD8␣␣ (crosses) decreased as the previous domain-swapping study demonstrated that the ␣3 concentration of ILTs increased, indicating that binding was not domain is required for ILT binding (17). Taken together these additive and that ILTs inhibited CD8␣␣ binding to these MHCIs. data suggest that the ILT-binding site overlaps with the portion In assays in which the concentration of ILT used was saturating, of the CD8␣␣-binding site located on the ␣3 domain. Consistent this inhibition was complete (Fig. 2 F–I), which is typical of with this observation, ILT4 binding to HLA-Cw7 and -G1 was competitive inhibition and indicates that the ILT and CD8␣␣- not affected by changing the peptide in the peptide groove (data binding sites on MHCI overlap or are close enough to each other not shown). to block the binding. This is consistent with the demonstration To localize the ILT-binding site on MHCIs further, we took that ILT binding requires the ␣3 domain of MHCIs (17). advantage of our finding that ILTs bind to HLA-G1 with a We next examined the effect of the anti-␤2m mAb BBM.1 on significantly higher affinity than to classical MHCIs and the fact that the binding of ILTs and CD8␣␣ (Fig. 2J). The binding responses these differences are likely to result from a relatively small number observed for ILT2, ILT4, and CD8␣␣ to three different MHCIs of sequence differences (Fig. 3A). The regions on the surface of were the same as before and after saturating the MHCI with MHCIs that differ between HLA-G1 and the classical MHCIs are BBM.1 mAb (Fig. 2J), indicating that the BBM.1-binding site shown in Fig. 3C (orange and green surfaces on Upper Left and does not overlap with either the CD8␣␣- or ILT-binding sites. Upper Right, respectively). They cluster in two sites: (i) the G strand There is some controversy as to the exact position of the residues 268, 271, 275, and 276 (green in Fig. 3C Upper Right) and BBM.1-binding site on ␤2m; one putative site overlaps with the (ii) the CCЈE ␤-strand residues 195, 197, 214, and 228 (orange in CD8␣␣ site (residues 58–63 and 91–95; shown in cyan on ␤2m Fig. 3C Upper Left). Given that the CD8␣␣- and ILT-binding sites in Fig. 3C) (33), whereas the other proposed site (residues 38, 44, overlap, it is unlikely that site 1 residues (green) contribute to ILT

and 45; blue surfaces in Fig. 3C) does not (34). Our results binding. However, site 2 residues (orange) are well positioned to IMMUNOLOGY suggest that the latter site is the BBM.1 epitope. contribute to ILT binding (Fig. 3C).

Shiroishi et al. PNAS ͉ July 22, 2003 ͉ vol. 100 ͉ no. 15 ͉ 8859 Downloaded by guest on September 25, 2021 Fig. 3. The putative ILT-binding site of MHCI. (A) Amino acid sequence alignment of ␣3 domains of HLA class I alleles (183–276). (B) Amino acid sequence alignment of the D1 and D2 domains of ILT2 and ILT4. (C Upper Left) Surface and ribbon diagram of HLA class I molecule with solid (␣3 domain) and transparent surface. The putative residues playing an important role in strong binding of ILT2 and ILT4 to HLA-G1 (see Results and Discussion) are shown in orange. The residues of the ␣3 domain involved in MHCI–CD8␣␣ binding are shown in cyan (35). The putative residues playing a part in the differentiation of ILT binding to different MHCI alleles are shown in pink. The residues interacting with KIR2DL1 are shown in red. The important epitopes of anti-␤2m antibody BBM.1 (residues 38, 44, and 45) are shown in blue (34). (Upper Right) The reverse side view of that shown in the Upper Left. The residues of HLA-G1, which differ from those of the other MHCI alleles on the G strand, are shown in green. (Lower) Surface and ribbon diagram of ILT2D1D2. The residues playing an important role in binding to UL18 are shown in yellow (18). The colors used in this figure correspond to those of alignment shown in A and B. The diagrams in C were created with WEBLAB VIEWER LITE (Accelrys, San Diego).

There is some evidence that the ILT2- and ILT4-binding sites are to ILT2. Second, Allen et al. (36) reported that the free form of not identical. First, as shown in Table 1, HLA-Cw7 produced by HLA-B27 lacking ␤2m bound ILT4-transfected but not ILT2- using the chemically biotinylated ␤2m could bind to ILT4 but not transfected cells. These results suggest that ILT2 binding depends more on ␤2m than ILT4 binding. Perhaps the ILT2-binding site incorporates the region around the ␣3–␤2m interface. Table 1. Summary of affinity constants of the interactions of Our results indicate that, although both ILT2 and ILT4 bind ILT2D1D2 and ILT4D1D2 to MHCIs to a range of MHCIs, there are significant variations in the binding affinities. On the one hand, ILTs bind HLA-G with a Injected, Kd at 25°C, ␮M higher affinity than they bind classic MHCIs. One notable Immobilized sILT2D1D2 sILT4D1D2 CD8␣␣ difference in the putative ILT-binding site is the relatively ͞ HLA-A11 ND 45 Ϯ 17 (3) ND hydrophobic patch formed by F195 Y197 on HLA-G, which is HLA-B35 8.8 Ϯ 0.2 (5) 26 Ϯ 4.6 (8) 126 Ϯ 3 (4) absent in other MHCs where the corresponding residues are HLA-Cw4 6.5 Ϯ 0.5 (5) 14 Ϯ 2.0 (4) 210 Ϯ 10 (2) S195͞H197. On the other hand, ILT2 has a higher affinity and HLA-G1 2.0 Ϯ 0.7 (11) 4.8 Ϯ 1.4 (10) 72 Ϯ 1.4 (4) narrower specificity than ILT4. What might be the structural HLA-Cw7/DS11* NB 26 Ϯ 6.0 (8) ND basis for this difference? Comparison of sequences in the HLA-Cw7/DS12* NB 23 Ϯ 6.2 (4) ND putative MHCI-binding site of ILT molecules indicates notable Shown is the mean Ϯ standard deviation. The number of measurements is differences (Fig. 3B). For example the putative binding region on shown in parentheses. The immobilized levels of MHCs are from 800 to 3,000 ILT2D1D2 (18) includes residues (Y76, D80, and R84) that are response units. NB, no binding observed at the ILT2D1D2 concentration of 87 not conserved in ILT4 (Q76, R80, and R84) (Fig. 3C). Further- ␮M; ND, not determined. more, there is evidence, noted above, that ILT2 but not ILT4 *Chemically biotinylated ␤2m was used. binding to MHCI depends on ␤2m, suggesting a difference in the

8860 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1431057100 Shiroishi et al. Downloaded by guest on September 25, 2021 ILT2- versus ILT4-binding sites on MHCI. Thus there is signif- modulated in some way. One possibility is that CD8 is effectively icant variation in the ILT–MHCI binding interfaces that could recruited to the TCR–CD3 complex, thereby enhancing its easily account for the observed variation in binding affinities. ability to engage MHCIs. Whether these differences are physiologically important remains The relatively high affinity of ILT2 binding to HLA-G is con- to be shown. sistent with previous observations that the effects of ILT2-mediated inhibition on peripheral blood NK cells can be largely attributed to Functional Implications. Our finding that ILT2 and ILT4 bind with HLA-G1 recognition (38). Similarly, the ILT2–HLA-G interaction a higher affinity to HLA-G than to classical MHCIs raises the may also effectively inhibit NK cell recognition of trophoblast and possibility that both the ILTs may contribute to functional HLA-G-expressing tumor cells, thereby contributing to materno- interactions between leukocytes expressing ILTs (T, NK, and fetal tolerance and escape of tumor cells. myelomonocytic cells) and cells expressing HLA-G. The latter Although the affinity of ILT4 binding to HLA-G is slightly include thymic epithelial cells, fetal trophoblast tissue, some lower than ILT2, ILT4 shows a much stronger preference for cancers, and the cells infected by human cytomegalovirus (37). HLA-G versus classical MHCIs than does ILT2, which suggests ILT2 may influence thymocyte development by interacting that the ILT4–HLA-G interaction may be of considerable with HLA-G and classical MHCIs on thymic epithelial cells, significance for regulating the maturation and͞or function of thereby modulating the threshold of TCR triggering. It is cells in the myelomonocytic lineage. Indeed, it has been sug- noteworthy that the binding affinity of the ILT2–HLA-G inter- gested that ILT4 also may regulate the threshold of myelomono- action is at the high end of the range of affinities measured for cytic activation in materno-fetal tolerance (12), inflammatory ϭ ␮ TCR–MHCs interactions (Kd 1–50 M). responses (11), and tolerogenic antigen-presenting cells induced We also show that ILTs compete directly with CD8␣␣ for by regulatory T cells (13). binding to MHCIs. Whereas intraepithelial T cells express In conclusion, we report here that ILT2 and ILT4 bind more CD8␣␣, most T cells express CD8␣␤. However, our results are strongly to HLA-G than to classical MHCIs, that ILT2 binds with likely to be relevant for all T cells, because CD8␣␣ and CD8␣␤ a higher affinity than ILT4, and that ILTs compete with CD8 for have comparable affinities for MHC, and these affinities are binding to MHCIs. These observations provide insights into the considerably lower than the affinity of ILT2 binding to MHCI. possible role of ILT–MHCI interactions in regulating immuno- The higher affinity of ILT versus CD8 binding suggest that ILTs logical recognition. may effectively block CD8 binding at the cell–cell interface. Interestingly, Dietrich et al. (9) showed that ILT2 and TCR Note Added in Proof. The conclusion that site 2 residues are well colocalize at the immunological synapse formed between T cells positioned to contribute to ILT binding is supported by a recently and antigen-presenting cells expressing ligands for ILT2 (HLA- determined crystal structure of LIR-1 (ILT2) bound to a classical MHCI B27) and TCR (superantigen) on their surface. These data (B.W., L. M. Thomas, and P. J. Bjorkman, unpublished data). suggested that ILT2 could potentially function as an ‘‘inhibitory’’ coreceptor, first by blocking binding of CD8 and second by We thank D. I. Stuart and G. F. Gao for discussion; L. Lanier for the bringing immunoreceptor tyrosine-based inhibitory receptor 40H2 mAb; and E. Davies for assistance. We also thank L. M. Thomas and P. J. Bjorkman for giving us the unpublished information of the motifs with their associated tyrosine phosphatases into proximity ILT–MHC complex structure. K.M. was supported in part by the with the TCR engaging the same peptide-MHCI. There is at Ministry of Education, Science, Sports, Culture, and Technology of present no direct evidence for a ‘‘competitive’’ inhibitory effect, Japan, the 2000th year Joint Research Project (Soken͞K01-4) of Sok- ϩ and the fact that CD8 T cells that express ILT2 can be activated endai, and a research grant from the Nakajima foundation. E.Y.J. is by recognition of MHCIs suggests that this competition may be supported by Cancer Research U.K. and the Medical Research Council.

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